Resistance to the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has been attributed solely to mutations in BTK and related pathway molecules. Using whole-exome and deep-targeted sequencing, we dissect evolution of ibrutinib resistance in serial samples from five chronic lymphocytic leukaemia patients. In two patients, we detect BTK-C481S mutation or multiple PLCG2 mutations. The other three patients exhibit an expansion of clones harbouring del(8p) with additional driver mutations (EP300, MLL2 and EIF2A), with one patient developing trans-differentiation into CD19-negative histiocytic sarcoma. Using droplet-microfluidic technology and growth kinetic analyses, we demonstrate the presence of ibrutinib-resistant subclones and estimate subclone size before treatment initiation. Haploinsufficiency of TRAIL-R, a consequence of del(8p), results in TRAIL insensitivity, which may contribute to ibrutinib resistance. These findings demonstrate that the ibrutinib therapy favours selection and expansion of rare subclones already present before ibrutinib treatment, and provide insight into the heterogeneity of genetic changes associated with ibrutinib resistance.

Circulating tumor cells (CTCs) are shed into the bloodstream by invasive cancers, but the difficulty inherent in identifying these rare cells by microscopy has precluded their routine use in monitoring or screening for cancer. We recently described a high-throughput microfluidic CTC-iChip, which efficiently depletes hematopoietic cells from blood specimens and enriches for CTCs with well-preserved RNA. Application of RNA-based digital PCR to detect CTC-derived signatures may thus enable highly accurate tissue lineage-based cancer detection in blood specimens. As proof of principle, we examined hepatocellular carcinoma (HCC), a cancer that is derived from liver cells bearing a unique gene expression profile. After identifying a digital signature of 10 liver-specific transcripts, we used a cross-validated logistic regression model to identify the presence of HCC-derived CTCs in nine of 16 (56%) untreated patients with HCC versus one of 31 (3%) patients with nonmalignant liver disease at risk for developing HCC (P < 0.0001). Positive CTC scores declined in treated patients: Nine of 32 (28%) patients receiving therapy and only one of 15 (7%) patients who had undergone curative-intent ablation, surgery, or liver transplantation were positive. RNA-based digital CTC scoring was not correlated with the standard HCC serum protein marker alpha fetoprotein (P = 0.57). Modeling the sequential use of these two orthogonal markers for liver cancer screening in patients with high-risk cirrhosis generates positive and negative predictive values of 80% and 86%, respectively. Thus, digital RNA quantitation constitutes a sensitive and specific CTC readout, enabling high-throughput clinical applications, such as noninvasive screening for HCC in populations where viral hepatitis and cirrhosis are prevalent.circulating tumor cells | early cancer detection | hepatocellular carcinoma | blood biopsy | predictive modeling T he shedding by epithelial cancers of circulating tumor cells (CTCs) into the bloodstream underlies the blood-borne dissemination of cancer, although only a small fraction of CTCs gives rise to metastases (1). Enumeration and analysis of CTCs may thus enable noninvasive monitoring of advanced cancers, as well as early detection of invasive but localized tumors before they give rise to viable metastases. Recent advances in CTC isolation provide sensitive and high-throughput platforms to enrich for these rare tumor cells within blood specimens, but antibody staining and microscopic imaging of captured cancer cells remain a critical bottleneck limiting broad application of the technology (2). Classical CTC staining criteria include the presence of cell surface epithelial cell adhesion molecule (EpCAM) and cytoplasmic epithelial cytokeratins and the absence of the hematopoietic CD45 marker (3), but epithelial marker expression is highly variable and extensive imaging criteria must be applied to score immunofluorescent signals reliably from rare cancer cells surrounded by contaminating leukocytes (4). Emerging microfluidic C...

Garlic polysaccharides are great potential agents because of their anti-inflammation, antioxidation, and immunomodulation properties. However, few studies have reported their anti-inflammatory effects on improving the colon system and corresponding intestinal microbiota. Herein, a water-soluble garlic polysaccharide (WSGP) was extracted from Jinxiang garlic to evaluate its effects on ameliorating dextran sulfate sodium (DSS)-induced colitis in a mouse model. The results showed that (1) after administration of the WSGP (200 or 400 mg/kg/day), the feed intake, body weight, and colon length of colitic mice were increased, while the disease activity index and the histological score of colitic mice were decreased; (2) the WSGP reduced the colonic tissue damage and inhibited the expression of inflammatory factors (interleukin 6, interleukin 1 beta , and tumor necrosis factor alpha); and (3) the WSGP enhanced the production of short-chain fatty acids and improved the composition of intestinal microbiota. The key microorganisms, including Muribaculaceae, Lachnospiraceae, Lachnospiraceae_NK4A136_group, Mucispirillum, Helicobacter, Ruminococcus_1, and Ruminiclostridium_5, were identified to be associated with inflammatory bowel diseases. Taken together, this study proved that WSGP supplementation could alleviate DSS-induced colitis by improving mucosal barriers, blocking proinflammatory cytokines, and modulating gut microbiota.

Wound healing is one of the most important and basic issues faced by the medical community. In this paper, we present biomass-composited inverse opal particles with a series of advanced features for drug delivery and wound healing. The particles were derived by using chitosan biomass to negatively replicate spherical colloid crystal templates. Because of the interconnected porous structures, various forms of active drugs, including fibroblast growth factor could be loaded into the void spaces of the inverse opal particles and encapsulated by temperature-responsive hydrogel. This endowed the composited particles with the capability of intelligent drug release through the relatively high temperature caused by the inflammation reaction at wound sites. Because the structural colors and characteristic reflection peaks of the composited inverse opal particles are blue-shifted during the release process, the drug delivery can be monitored in real time. It was demonstrated that the biomass-composited microcarriers were able to promote angiogenesis, collagen deposition, and granulation–tissue formation as well as reduce inflammation and thus significantly contributed to wound healing. These features point to the potential value of multifunctional biomass inverse opal particles in biomedicine.

Embryonic hematopoiesis is a complex process. Elucidating the mechanism regulating hematopoietic differentiation from pluripotent stem cells would allow us to establish a strategy to efficiently generate hematopoietic cells. However, the mechanism governing the generation of hematopoietic progenitors from human embryonic stem cells (hESCs) remains unknown. Here, on the basis of the emergence of CD43 + hematopoietic cells from hemogenic endothelial (HE) cells, we demonstrated that VEGF was essential and sufficient, and that bFGF was synergistic with VEGF to specify the HE cells and the subsequent transition into CD43 + hematopoietic cells. Significantly, we identified TGFβ as a novel signal to regulate hematopoietic development, as the TGFβ inhibitor SB 431542 significantly promoted the transition from HE cells into CD43 + hematopoietic progenitor cells (HPCs) during hESC differentiation. By defining these critical signaling factors during hematopoietic differentiation, we can efficiently generate HPCs from hESCs. Our strategy could offer an in vitro model to study early human hematopoietic development.

Correlative light and electron microscopy promises to combine molecular specificity with nanoscale imaging resolution. However, there are substantial technical challenges including reliable co-registration of optical and electron images, and rapid optical signal degradation under electron beam irradiation. Here, we introduce a new approach to solve these problems: imaging of stable optical cathodoluminescence emitted in a scanning electron microscope by nanoparticles with controllable surface chemistry. We demonstrate well-correlated cathodoluminescence and secondary electron images using three species of semiconductor nanoparticles that contain defects providing stable, spectrally-distinguishable cathodoluminescence. We also demonstrate reliable surface functionalization of the particles. The results pave the way for the use of such nanoparticles for targeted labeling of surfaces to provide nanoscale mapping of molecular composition, indicated by cathodoluminescence colour, simultaneously acquired with structural electron images in a single instrument.

Niacin metal-organic frameworks (MOFs) encapsulated microcapsules with alginate shells and copper-/zinc-niacin framework cores were in situ synthesized by using a microfluidic electrospray approach for wound healing. As the alginate shells were bacteria-responsively degradable, the niacin MOFs encapsulated microcapsules could intelligently, controllably, and programmably release calcium, copper, and zinc ions, depending on the degree of infections. The released ions could not only kill microbes by destroying their membrane and inducing the outflow of nutrient substance, but also activate copper/zinc superoxide dismutase (Cu/Zn-SOD) to eliminate oxygen free radicals and rescue the cells from oxidative stress injury. Furthermore, the simultaneously released niacin could promote hemangiectasis and absorption of functional metal ions. Thus, the niacin MOFs encapsulated microcapsules were imparted with outstanding antibacterial, antioxidant, and angiogenesis properties. Based on an in vivo study, we have also demonstrated that the chronic wound healing process of an infected full-thickness skin defect model could be significantly enhanced by using the niacin MOFs encapsulated microcapsules as therapeutic agent. Therefore, the microfluidic electrospray niacin MOFs encapsulated microcapsules are potential for clinical applications.

A key viral property is infectivity, and its accurate measurement is crucial for the understanding of viral evolution, disease and treatment. Currently viral infectivity is measured using plaque assays, which involve prolonged culturing of host cells, and whose measurement is unable to differentiate between specific strains and is prone to low number fluctuation. We developed a rapid, targeted and culture-free infectivity assay using high-throughput drop-based microfluidics. Single infectious viruses are incubated in a large number of picoliter drops with host cells for one viral replication cycle followed by in-drop gene-specific amplification to detect infection events. Using murine noroviruses (MNV) as a model system, we measure their infectivity and determine the efficacy of a neutralizing antibody for different variants of MNV. Our results are comparable to traditional plaque-based assays and plaque reduction neutralization tests. However, the fast, low-cost, highly accurate genomic-based assay promises to be a superior method for drug screening and isolation of resistant viral strains. Moreover our technique can be adapted to measuring the infectivity of other pathogens, such as bacteria and fungi.